[Truncated abstract] Calcium and reactive oxygen species have been demonstrated to participate as partners in the progression toward cardiac hypertrophy. However the early mechanisms associated with progression toward cardiac hypertrophy are poorly understood. A feedback mechanism which may have the potential to mediate cardiac pathology involving increased L-type Ca2+ channel activity, intracellular calcium and mitochondrial superoxide production in response to exposure of cardiac myocytes to 30 μmol/l H2O2 for 5 min has been proposed. This transient H2O2 exposure was intended to mimic the effect of transient oxidative stress in vivo in humans associated with ischemia-reperfusion injury. This thesis investigates the early mechanisms that lead to the development of pathological remodeling and progression toward cardiac hypertrophy in vitro in response to mild oxidative stress, by examining the mechanisms by which the L-type Ca2+ channel regulates mitochondrial function in the heart. The site of superoxide production within guinea pig ventricular myocyte mitochondria in response to transient H2O2 exposure was investigated. Complex I inhibitors (DPI and rotenone) and inhibitors of the Qo site of complex III (myxothiazol and stigmatellin) attenuated the increase in superoxide after transient H2O2 exposure, but antimycin A, an inhibitor of Qi site of complex III, had no effect. This is evidence that elevated superoxide production after transient H2O2 exposure occurs at the Qo site of complex III. A novel experimental design was used to "isolate" the Qo site of complex III in intact myocytes. Under these conditions, myxothiazol attenuated the increase in superoxide, confirming that elevated superoxide production after transient H2O2 exposure occurs at the Qo site of complex III. TCA cycle inhibitors KMV and 4-HNE also attenuated the increase in superoxide. This is evidence that an increase in TCA cycle activity plays a significant role in mediating the response. Finally, intracellular application of catalase attenuated the increase in superoxide. This indicates that H2O2 is the signal responsible for feeding back on to the L-type Ca2+ channel resulting in sustained activation of the channel. Studies were performed to determine if transient H2O2 exposure is sufficient to alter protein expression and function in the myocytes. .... Studies performed in ventricular myocytes isolated from dystrophin-deficient (mdx) mice consolidate these findings, since activation of the L-type Ca2+ channel in the absence of calcium caused a significant increase in Ψm and metabolic activity in wild-type but not mdx mice. Determining the early mechanisms associated with progression toward cardiac hypertrophy is critical to the development of interventions to prevent heart failure. This thesis examines the mechanisms by which the L-type Ca2+ channel regulates mitochondrial function in the heart, providing insight into the early mechanisms that lead to the development of pathological remodeling and progression toward cardiac hypertrophy in vitro in response to mild oxidative stress. In light of the data presented in this thesis, the L-type Ca2+ channel, TCA cycle and Qo site of complex III may represent possible sites to target in the prevention of progression toward cardiac hypertrophy associated with mild oxidative stress.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2010|